Dye-binding Assay Research Articles

Pigment Binding, Fluorescence Properties, and Oligomerization Behavior of Lhca5, a Novel Light-harvesting Protein

A new potential light-harvesting protein, named Lhca5, was recently detected in higher plants. Because of the low amount of Lhca5 in thylakoid membranes, the isolation of a native Lhca5 pigment-protein complex has not been achieved to date. Therefore, we used in vitro reconstitution to analyze whether Lhca5 binds pigments and is actually an additional light-harvesting protein. By this approach we could demonstrate that Lhca5 binds pigments in a unique stoichiometry. Analyses of pigment requirements for light-harvesting complex formation by Lhca5 revealed that chlorophyll b is the only indispensable pigment. Fluorescence measurements showed that ligated chlorophylls and carotenoids are arranged in a way that allows directed energy transfer within the light-harvesting complex. Reconstitutions of Lhca5 together with other Lhca proteins resulted in the formation of heterodimers with Lhca1. This result demonstrates that Lhca5 is indeed a protein belonging to the light-harvesting antenna of photosystem I. The properties of Lhca5 are compared with those of previously characterized Lhca proteins, and the consequences of an additional Lhca protein for the composition of the light-harvesting antenna of photosystem I are discussed in view of the recently published photosystem I structure of the pea.

Alteration of Lithium Pharmacology through Manipulation of Phosphoadenosine Phosphate Metabolism

Bisphosphate 3'-nucleotidase (BPNT1 in mammals and Met22/Hal2 in yeast) is one of five members of a family of signaling phosphatases united through a common tertiary structure and inhibition by subtherapeutic doses of the antibipolar drug lithium. Here we report a role for 3'-nucleotidase and its substrate, 3'-phosphoadenosine 5'-phosphate (PAP), in mediating the cellular effects of lithium. Lithium-induced inhibition of growth in yeast cells may be overcome by dose-dependent heterologous expression of human BPNT1. Disruption of the yeast 3'-nucleotidase gene or treatment of cells with lithium results in a >80-fold accumulation of PAP and leads to potent growth inhibition. These data indicate that the accumulation of a 3'-nucleotidase substrate, such as PAP, mediates the toxicity of lithium. To further probe this model we examined the growth inhibitory effects of lithium under conditions in which PAP biosynthetic machinery was concomitantly down-regulated. Disruption of met3 or met14 genes (ATP sulfurylase or phosphosulfate kinase), transcriptional down-regulation of MET3 through methionine addition, or administration of chlorate, a widely used cell-permeable sulfurylase inhibitor, function to reduce lithium-induced intracellular PAP accumulation and lithium toxicity; all of these effects were reversed by heterologous expression of human sulfurylase and kinase. Collectively, our data support a role for 3'-nucleotidase activity and PAP metabolism in aspects of lithium's mechanism of action and provide a platform for development of novel pharmacological modulators aimed at improving therapies for the treatment of bipolar disorder.

Cartilage-derived morphogenetic protein-1 promotes the differentiation of mesenchymal stem cells into chondrocytes

Mesenchymal stem cells (MSCs) are able to differentiate into many types of cells including chondrocytes. Transforming growth factor β1 (TGF-β1) is very important in the regulation of chondrogenesis. Since cartilage-derived morphogenetic protein-1 (CDMP-1) belongs to the TGF-β superfamily, we tested whether CDMP-1 plays any role in the regulation of the differentiation of MSCs into chondrocytes using a high density pellet culture system. Based on the histological staining of glycosaminoglycan using toluidine blue dye-binding method we found that CDMP-1 could initiate chondrogenic differentiation of MSCs as did TGF-β1. However, CDMP-1 was less stimulatory than TGF-β1. The combination of CDMP-1 and TGF-β1 synergically induced chondrogenesis of MSCs. This synergic chondrogenic effect of CDMP-1 together with TGF-β1 was further confirmed by quantification of GAG using dimethylmethylene blue dye-binding assay and immunohistochemical analysis of the expression of cartilage-specific protein collagen II. This study may provide an improved induction approach using MSCs for repairing damaged cartilage.

A new interferon, limitin, displays equivalent immunomodulatory and antitumor activities without myelosuppressive properties as compared with interferon-α

Limitin is a new member of type I interferon (IFN) identified with an expression cloning based on the growth suppression of a myelomonocytic leukemia cell line WEHI3. Although limitin uses the IFN-alpha/beta receptor, its signal transduction pathways to express the antiviral effects are different from those of IFN-alpha. To clarify the characteristics of limitin, we compared the biological activities of limitin, such as the antiviral, immunomodulatory, antitumor, and myelosuppressive effects, with IFN-alpha. Limitin and IFN-alpha were titered with a cytopathic effect dye binding assay. Induction of MHC class I on a keratinocyte cell line PAM212 was estimated with flow cytometry. Induction of OVA-restricted cytotoxic T lymphocyte (CTL) activity was analyzed with 51Cr release assay. Antiproliferative effects were evaluated with 3H-thymidine incorporation assay using WEHI3 and a lymphoblast cell line L1210. Myelosuppresive effects were evaluated with colony assay. In vivo side effects were estimated after the injection of limitin or IFN-alpha. Limitin had relatively higher antiviral activity than IFN-alpha. Limitin induced the surface expression of MHC class I, the enhancement of CTL activity, and the growth inhibition of lymphohematopoietic cell lines as strong as IFN-alpha. Nevertheless, the treatment of mice with limitin showed neither myelosuppression nor fever that are common adverse effects of IFN-alpha. Strong immunomodulatory, antitumor, and antiviral effects with weak myelosuppressive and weak acute toxic effects of limitin indicate that it may be useful as a new therapeutic drug for virus-hepatitis and cancers.

Elimination of the interference from aminoglycoside antibiotics in the pyrogallol red-molybdate protein dye-binding assay.

The Pyrogallol Red-molybdate (PRM) protein dye-binding assay is used for urinary protein determination (1)(2)(3). The assay is sensitive to interference from aminoglycoside antibiotics, and the extent of interference varies with different PRM reagents (1)(2)(4)(5)(6)(7). Thus, the Dade Behring PRM and the Sigma PRM assays are sensitive to aminoglycoside interference, whereas the Cobas Fara and Roche Integra 700 PRM assays are not (4)(5). Furthermore, the PRM reagents used by Fujita et al. (1) and Orsonneau et al. (3) are more sensitive to interference than the reagent used by Watanabe et al. (2). We have previously shown that the addition of sodium dodecyl sulfate (SDS) to the PRM reagent increases the interference, whereas the addition of sodium oxalate reduces the interference (6)(7). In the present …

Pressure activates rat pancreatic stellate cells.

Pancreatic stellate cells (PSCs) play a central role in development of pancreatic fibrosis. In chronic pancreatitis, pancreatic tissue pressure is higher than that of the normal pancreas. We here evaluate the effects of pressure on the activation of rat PSCs. PSCs were isolated from the pancreas of Wistar rat using collagenase digestion and centrifugation with Nycodenz gradient. Pressure was applied to cultured rat PSCs by adding compressed helium gas into the pressure-loading apparatus to raise the internal pressure. Cell proliferation rate was assessed by 5-bromo-2'-deoxyuridine (BrdU) incorporation. MAPK protein levels and alpha-smooth muscle actin (alpha-SMA) expression were evaluated by Western blot analysis. Concentration of activated transforming growth factor-beta1 (TGF-beta1) secreted from PSCs into culture medium was determined by ELISA. Collagen type I mRNA expression and collagen secretion were assessed by quantitative PCR and Sirius red dye binding assay, respectively. Application of pressure significantly increased BrdU incorporation and alpha-SMA expression. In addition, pressure rapidly increased the phosphorylation of p44/42 and p38 MAPK. Treatment of PSCs with an MEK inhibitor and p38 MAPK inhibitor suppressed pressure-induced cell proliferation and alpha-SMA expression, respectively. Moreover, pressure significantly promoted activated TGF-beta1 secretion, collagen type I mRNA expression, and collagen secretion. Our results demonstrate that pressure itself activates rat PSCs and suggest that increased pancreatic tissue pressure may accelerate the development of pancreatic fibrosis in chronic pancreatitis.

The role of insulin-like growth factor-I in hyaluronan mediated repair of cultured cartilage explants.

To test whether hyaluronan (HA) restores proteoglycan (PG) in fibronectin fragment (Fn-f) damaged cartilage through activities of insulin-like growth factor 1 (IGF-1). Bovine cartilage explants were studied. Cartilage PG content was measured by a dye binding assay. PG synthesis was measured by incorporation of radiolabeled sulfate. Release of IGF-1 from explants was measured by ELISA. Higher mass HA forms were more reparative than smaller and enhanced PG synthesis in explants, but not in monolayer cultures. Higher mass HA enhanced PG synthesis and retention of IGF-1 within the explants while IGF-1 binding proteins decreased activities of HA. The reparative effects of HA appear to involve enhanced PG synthesis through IGF-1 and requires cartilage matrix.

Fibronectin fragments elevate nitric oxide (NO) and inducible NO synthetase (iNOS) levels in bovine cartilage and iNOS inhibitors block fibronectin fragment mediated damage and promote repair.

To determine whether fibronectin fragments (Fn-f) upregulate nitric oxide (NO) and inducible NO synthetase (iNOS) levels in explants and whether iNOS inhibitors block Fn-f mediated cartilage damage or promote repair. Bovine cartilage explants were studied. Explants were cultured with Fn-f and involvement of NO pathway investigated. Induction of iNOS was tested by RT-PCR. Upregulation of iNOS protein was visualized by Western blotting. Proteoglycan (PG) content of cartilage treated with iNOS inhibitors was tested by dye binding assays of cartilage digests. Fn-fs elevated NO release in explants and upregulated iNOS protein and mRNA while native Fn had only weak activity. Three different iNOS inhibitors blocked Fn-f mediated PG depletion from explants and facilitated restoration of PG in pre-damaged cartilage. The Fn-f cartilage damaging pathway involves upregulation of iNOS. Interestingly, iNOS inhibitors not only blocked damage but repaired damaged cartilage. This may be the first report of reparative activities of iNOS inhibitors.

Temporal expression of growth factors and matrix molecules in healing tendon lesions

Overuse tendon injuries are common among elite and recreational athletes. Tendon healing may be enhanced at the cellular level through the use of exogenous growth factors; however, little is known about the endogenous expression of growth factors in healing tendon. This study describes the temporal expression of insulin-like growth factor-I (IGF-I), transforming growth factor-β1 (TGF-β1), and collagen types I and III in healing tendon lesions. Collagenase-induced lesions were created in the tensile region of the flexor digitorum superficialis tendon of both forelimbs of 14 horses. Tendons were harvested from euthanatized horses 1, 2, 4, 8 or 24 weeks following injury. Gene expression was evaluated using Northern blot analysis (collagen types I and III), real time PCR (IGF-I and TGF-β1), and in situ hybridization. Protein content was assayed by dye-binding assay (collagen types I and III), radioimmunoassay (IGF-I), ELISA (TGF-β1), and immunohistochemistry. Samples were also processed for differential collagen typing, DNA and glycosaminoglycan content, and routine H&E staining. Microscopically, lesions progressed from an amorphous, acellular lesion soon after injury to scar tissue filled with collagen fibers and mature fibroblasts organized along lines of tension. Early lesions were characterized by immediate increases in expression of growth factors and collagen. Message levels for TGF-β1 peaked early in the wound healing process (1 week), while IGF-I peaked later (4 weeks), as the regenerative phase of healing was progressing. In the first 2 weeks after lesion induction, tissue levels of IGF-I protein actually decreased approximately 40% compared to normal tendon. By 4 weeks, these levels had exceeded those of normal tendon and remained elevated through 8 weeks. Message expression for collagen types I and III increased by 1 week following injury and remained elevated throughout the course of the study. Collagen type I represented the major type of collagen in healing tendon at all time points of the study. Based on these results, IGF-I, administered exogenously during the first 2 weeks following injury, may provide a therapeutic advantage by bolstering low endogenous tissue levels and enhancing the metabolic response of individual tendon fibroblasts.

Interference in the Coomassie Brilliant Blue and Pyrogallol Red protein dye-binding assays is increased by the addition of sodium dodecyl sulfate to the dye reagents

We have investigated the effect of sodium dodecyl sulfate (SDS) upon the response of the Coomassie Brilliant Blue (CBB) and Pyrogallol Red-molybdate (PRM) protein dye-binding assays to interference from aminoglycosides, ampholytes, detergents, phenothiazines, reducing agents, and miscellaneous substances previously reported to interfere with the assays. The CBB assay was less prone to interference than the PRM assay but gave positive interference with the detergents and the phenothiazines and negative interference with dextran sulfate. The PRM assay gave positive interference with the aminoglycosides, ampholytes, and phenothiazines and negative interference with SDS, citric acid, dextran sulfate, EDTA, oxalic acid, and tartaric acid. The level of interference varied in the presence of different proteins (albumin, gamma globulin, α 1-acid glycoprotein, or lysozyme) and increased when SDS was added to the dye reagents.

Effects of growth factors on temporomandibular joint disc cells

The effects of growth factors on cartilaginous tissues are well documented. An exception is the temporomandibular joint (TMJ) disc, where data for growth factor effects on proliferation and biosynthesis are very limited. The purpose of this study was to quantify proliferation of and synthesis by TMJ disc cells cultured in monolayer with either platelet derived growth factor-AB (PDGF), basic fibroblast growth factor (bFGF) or insulin-like growth factor-I (IGF), at either a low (10 ng/ml) or high (100 ng/ml) concentration. Proliferation was assessed with a DNA quantitation technique, collagen synthesis was measured via a hydroxyproline assay, and GAG synthesis was determined with a dimethylmethylene blue dye binding assay at 14 days. Overall, the most beneficial growth factor was bFGF, which was most potent in increasing proliferation and GAG synthesis, and also effective in promoting collagen synthesis. At the high concentration, bFGF resulted in 96% more cells than the control and 30 to 45% more cells than PDGF and IGF. PDGF and bFGF were the most potent upregulators of GAG synthesis, producing 2–3 times more GAG than the control. IGF had no significant effect on GAG production, although at its higher concentration it increased collagen production by 4.5 times over the control. Collagen synthesis was promoted by bFGF at its lower concentration, with levels 4.2 times higher than the control, whereas PDGF had no significant effect on collagen production. In general, higher concentrations increased proliferation, whereas lower concentrations favoured biosynthesis.

Amyloid Fibril Formation by Lens Crystallin Proteins and Its Implications for Cataract Formation

The alpha-, beta-, and gamma-crystallins are the major structural proteins within the eye lens and are responsible for its exceptional stability and transparency. Under mildly denaturing conditions, all three types of bovine crystallin assemble into fibrillar structures in vitro. Characterization by transmission electron microscopy, dye binding assays, and x-ray fiber diffraction shows that these species have all of the characteristics of fibrils associated with the family of amyloid diseases. Moreover, the full-length proteins are incorporated into the fibrils, (i.e. no protein cleavage is required for these species to form), although for the gamma-crystallins some fragmentation occurs under the conditions employed in this study. Our findings indicate that the inherent stability of the beta-sheet supramolecular structure adopted by the crystallins in the eye lens and the chaperone ability of alpha-crystallin must be crucial for preventing fibril formation in vivo. The crystallins are very stable proteins but undergo extensive post-translational modification with age that leads to their destabilization. The ability of the crystallins to convert into fibrils under destabilizing conditions suggests that this process could contribute to the development of cataract with aging.

Determination of the total protein and triglyceride content of human breast milk on the Synchron CX7 Delta analyser.

Premature babies have improved clinical outcomes when fed breast milk with a relatively high protein content. Since there was no convenient way of measuring the macronutrition of breast milk we used our routine laboratory pyrogallol red dye binding method for cerebrospinal fluid microprotein (MTP) and our routine method for serum triglyceride to determine the total protein and triglyceride content of human breast milk. The total protein contents of whole and defatted breast milk samples, randomly collected from 115 nursing mothers, analysed using a pyrogallol red dye binding assay on a Synchron CX7 Delta analyser were compared with the total protein contents determined by dry combustion analysis. Triglyceride concentrations, determined on the Synchron CX7 Delta analyser were compared with their respective creamatocrits. Passing and Bablok regression analysis (95% confidence interval) gave the following regression equations: y = 5.98(5.48 to 6.56)x-1.32(-2.02 to -0.73) where y is whole milk MTP (g/L) and x is dry combustion analysis (g/100 g); y = 7.09 (6.54 to 7.78)x-2.44 (-3.46 to -1.67) where y is volume-corrected defatted milk MTP (g/L) and x is dry combustion analysis (g/100 g); y = 7.52 (6.86 to 8.24)x+0.90(-2.42 to 3.37) where y is whole milk triglyceride (mmol/L) and x is creamatocrit (%). The Synchron CX7 Delta total protein and triglyceride assays provide practical, rapid and reliable methods for the determination of the macronutrition in human milk.

A Fluorescent Probe-labeled Escherichia coli Aspartate Transcarbamoylase That Monitors the Allosteric Conformational State

A new system has been developed capable of monitoring conformational changes of the 240s loop of aspartate transcarbamoylase, which are tightly correlated with the quaternary structural transition, with high sensitivity in solution. Pyrene, a fluorescent probe, was conjugated to residue 241 in the 240s loop of aspartate transcarbamoylase to monitor changes in conformation by fluorescence spectroscopy. Pyrene maleimide was conjugated to a cysteine residue on the 240s loop of a previously constructed double catalytic chain mutant version of the enzyme, C47A/A241C. The pyrene-labeled enzyme undergoes the normal T to R structural transition, as demonstrated by small-angle x-ray scattering. Like the wild-type enzyme, the pyrene-labeled enzyme exhibits cooperativity toward aspartate, and is activated by ATP and inhibited by CTP at subsaturating concentrations of aspartate. The binding of the bisubstrate analogue N-(phosphonoacetyl)-l-aspartate (PALA), or the aspartate analogue succinate, in the presence of saturating carbamoyl phosphate, to the pyrenelabeled enzyme caused a sigmoidal change in the fluorescence emission. Saturation with ATP and CTP (in the presence of either subsaturating amounts of PALA or succinate and carbamoyl phosphate) caused a hyperbolic increase and decrease, respectively, in the fluorescence emission. The half-saturation values from the fluorescence saturation curves and kinetic saturation curves were, within error, identical. Fluorescence and small-angle x-ray scattering stopped-flow experiments, using aspartate and carbamoyl phosphate, confirm that the change in excimer fluorescence and the quaternary structure change correlate. These results in conjunction with previous studies suggest that the allosteric transition involves both global and local conformational changes and that the heterotropic effect of the nucleotides may be exerted through local conformational changes in the active site by directly influencing the conformation of the 240s loop.

PEPping Up the Calmodulin Response

Changes in intracellular calcium concentration [Ca 2+ ] i of varying amplitude, frequency, and duration provide a signaling code that can be deciphered through the action of calcium-binding proteins such as calmodulin (CaM). Putkey et al. investigated the role of PEP-19, a member of a class of highly abundant small, neuronal proteins with IQ motifs (a motif found in CaM-binding proteins) and no known catalytic function. They found that PEP-19 accelerated the rate of Ca 2+ association and dissociation from the CaM C domain, enabling this domain to respond to rapid fluctuations in [Ca 2+ ] i . The authors measured changes in the fluorescence of Quin-2, a Ca 2+ -indicator dye, and of tyrosine, an endogenous marker of Ca 2+ binding to the C domain, and discovered that PEP-19 accelerated Ca 2+ dissociation from the C domain of free CaM 40- to 50-fold (Ca 2+ dissociation from the N domain is too rapid to measure). The equilibrium binding affinity was unchanged, as determined by a competitive dye-binding assay, which indicated that the rate of Ca 2+ association was also increased; this was confirmed by measuring changes in tyrosine fluorescence. PEP-19 also greatly enhanced the rate of Ca 2+ dissociation from CaM bound to CaM-dependent protein kinase IIα. The authors used nuclear magnetic resonance to identify PEP-19 binding sites on the CaM C domain. Although binding to targets can modify CaM's Ca 2+ affinity (by slowing the dissociation rate), PEP-19 enhanced the rates of Ca 2+ association and dissociation, which enabled the C domain to respond to rapid Ca 2+ signals and helped fine-tune the response to the Ca 2+ signaling code. J. A. Putkey, Q. Kleerekoper, T. R. Gaertner, M. N. Waxham, A new role for IQ motif proteins in regulating calmodulin function. J. Biol. Chem . 278 , 49667-49670 (2003). [Abstract] [Full Text]

The Role of Hydrophobic Interactions in Amyloidogenesis: Example of Prion-Related Polypeptides

Conversion of the non-infectious, cellular forom of the prion protein (PrPC) to the infectious form (PrPSc) is thought to be driven by an α-helical to β-sheet conformational transition. To reveal the sequence determinants which encourage the transition to (β-fold, we study the synthetic peptides associated with hydrophobic conserved fragments of the N-terminal region of the prion protein. The structure of peptides in solution was probed under various thermodynamic conditions employing circular dichroism and sleady stale fluorescence spectroscopy as well as dye binding assays. The fluorescence methods utilized pyrene residues covalently attached to the end of the model peptides. In aqueous solutions, the structure assessments indicate the formation of metastable peptide aggregates; the molecular conformations within the peptide micelles are largely coiled. This stage in molecular assembly exists without significant β-strand formation, i.e., before the appearance of any ordered secondary structure delectable by circular dichroism. At moderate concentrations of trifluo-roethanol and/or acetonitrile, the conformational ensemble shifts towards β-strand formation, and the population of the amorphous aggregates decreases significantly. Overall, the present data indicate that hydrophobic interactions between side chains of the peptide variants prevent, in fact, the formation of the rigid (β-sheet structures. Encouragement of β-folds requires the deslabilizalion of local interactions in the peptide chain, which in vivo might be possible within cell membranes as well as within partly folded molecular forms

The Circularization of Amyloid Fibrils Formed by Apolipoprotein C-II

Amyloid fibrils have historically been characterized by diagnostic dye-binding assays, their fibrillar morphology, and a “cross- β” x-ray diffraction pattern. Whereas the latter demonstrates that amyloid fibrils have a common β-sheet core structure, they display a substantial degree of morphological variation. One striking example is the remarkable ability of human apolipoprotein C-II amyloid fibrils to circularize and form closed rings. Here we explore in detail the structure of apoC-II amyloid fibrils using electron microscopy, atomic force microscopy, and x-ray diffraction studies. Our results suggest a model for apoC-II fibrils as ribbons ∼2.1-nm thick and 13-nm wide with a helical repeat distance of 53 nm ± 12 nm. We propose that the ribbons are highly flexible with a persistence length of 36 nm. We use these observed biophysical properties to model the apoC-II amyloid fibrils either as wormlike chains or using a random-walk approach, and confirm that the probability of ring formation is critically dependent on the fibril flexibility. More generally, the ability of apoC-II fibrils to form rings also highlights the degree to which the common cross- β superstructure can, as a function of the protein constituent, give rise to great variation in the physical properties of amyloid fibrils.

Biochemical reference intervals for koi ( Cyprinus carpio )

Various biochemical parameters of koi were measured to construct biochemical reference intervals. Very low serum concentrations of creatinine, total bilirubin, uric acid and γ-glutamyl transferase activity were observed. Increased activities of creatine kinase, lactate dehydrogenase and aspartate aminotransferase were observed due to venepuncture through the skeletal muscle of the caudal peduncle; however, these changes were not statistically significant. Alanine aminotransferase and sorbitol dehydrogenase activities were affected the least, and these enzymes may be considered relatively liver specific in koi. Cellulose acetate serum protein electrophoresis also was performed to confirm the presence of a serum albumin-like protein in koi. However, the resultant protein fractions were highly variable and the albumin protein-like fraction did not correlate well with that determined by the BCG dye binding assay.

Effects of growth/differentiation factor-5 on human periodontal ligament cells.

Growth/differentiation factor-5 (GDF-5), a member of the transforming growth factor-beta superfamily, shows a close structural relationship to bone morphogenetic proteins and plays crucial roles in skeletal, tendon, and ligament morphogenesis. The mRNA encoding GDF-5 is also expressed during odontogenesis, especially in dental follicle tissue. While this suggests that GDF-5 participates in the formation of alveolar bone and the periodontal ligament, cementum, and dental root, the physiologic role of GDF-5 in these tissues in adulthood remains unclear. We therefore investigated GDF-5 effects upon cultures of human periodontal ligament (HPDL) cells. HPDL cells were obtained from healthy periodontal ligaments of individuals. Tetrazolium reduction assay was carried out for cell proliferation assay. Alkaline phosphatase (ALP) activity was estimated by measuring light absorbance at 405 nm. Reverse transcription-polymerase chain reaction (RT-PCR) and northern analysis were performed for gene expression in cultured HPDL cells. Sulfated glycosaminoglycan (sGAG) synthesis was evaluated by histochemical staining and a quantitative dye-binding method. Expression of GDF-5 and its receptor was demonstrated in HPDL cells by RT-PCR. ALP activity in HPDL cells was significantly decreased by addition of rhGDF-5 at 10-1000 ng/ml (p < 0.05). Although northern analysis showed little change in gene expression for collagen alpha2(I) in rhGDF-5-stimulated HPDL cells, rhGDF-5 dose-dependently enhanced cell proliferation. This proliferative effect persisted for 16 d. Alcian blue staining and dye-binding assays indicated that sGAG synthesis was enhanced by rhGDF-5. rhGDF-5 may provide an environment fostering periodontal healing or regeneration by affecting extracellular matrix metabolism.

Early Synergy between Aβ42 and Oxidatively Damaged Membranes in Promoting Amyloid Fibril Formation by Aβ40

Oxidative lipid membrane damage is known to promote the misfolding of Abeta42 into pathological beta structure. In fully developed senile plaques of Alzheimer's disease, however, it is the shorter and more soluble amyloid beta protein, Abeta40, that predominates. To investigate the role of oxidative membrane damage in the misfolding of Abeta40, we have examined its interaction with supported lipid monolayer membranes using internal reflection infrared spectroscopy. Oxidatively damaged lipids modestly increased Abeta40 accumulation, with adsorption kinetics and a conformation that are distinct from that of Abeta42. In stark contrast, pretreatment of oxidatively damaged monolayer membranes with Abeta42 vigorously promoted Abeta40 accumulation and misfolding. Pretreatment of saturated or undamaged membranes with Abeta42 had no such effect. Parallel studies of lipid bilayer vesicles using a dye binding assay to detect fibril formation and electron microscopy to examine morphology demonstrated that Abeta42 pretreatment of oxidatively damaged membranes promoted the formation of mature Abeta40 amyloid fibrils. We conclude that oxidative membrane damage and Abeta42 act synergistically at an early stage to promote fibril formation by Abeta40. This synergy could be detected within minutes using internal reflection spectroscopy, whereas a dye-binding assay required several days and much higher protein concentrations to demonstrate this synergy.